Solid supports containing immobilized molecules have been extensively used in research, in clinical analyses and in the commercial production of foods and chemicals (see e.g., U.S. Pat. No. 5.153,166, issued Oct. 6, 1992); Akashi et al., Bioconjugate Chem. 3:363-365 (1992)). Immobilized nucleic acids are used in hybridization assays (Lamture et al., Nucl. Acids Res. 22:2121-2125 (1994)) and immobilized proteins in radioimmuno or ELISA assays (see, U.S. Pat. No. 5,314,830, issued May 24, 1994). In addition, enzymes have been immobilized to facilitate their separation from product and to allow for their efficient and repetitive use.
There are a number of important considerations in the development of an effective immobilization procedure. First, the procedure must minimize non-specific adsorption of molecules. Ideally, it should result in a surface which binds nothing but the target of the immobilized molecule. Second, the procedure must maintain the functional integrity of the immobilized molecules. An immobilization procedure which destroys the ability of a ligand to recognize and bind its natural target is of little or no practical value. A third factor of importance is the stability of the bond between the support and the immobilized molecule. Non-covalently attached molecules tend to leach off supports during assays, thereby reducing accuracy and sensitivity. Finally, the efficiency of the procedure must be carefully considered. Inefficient coupling of molecules will reduce the cost effectiveness of a procedure and may result in a support with a low binding capacity for target molecules. Assays which utilize such supports are likely to have a poor sensitivity.
Traditional supports used for immobilizing nucleic acids include nitrocellulose membranes, nylon membranes, avidin modified beads, dextrans and polysaccharides. Carbodiimide has been used for immobilizing nucleic acids on paper, latex, glass, dextrans and polystyrene. In addition, bromoacetyl and thiol-derivatized oligonucleotides have been immobilized on sulfhydryl- and bromoacetyl-polyacrylamide supports.
Recent interest in the development of biosensors and bioelectronic devices has required the development of immobilization procedures which are compatible with materials that have not traditionally been used for immobilizing biomolecules (Lamture et al., Nucl. Acids Res. 22:2121-2125 (1994)). These procedures must result in devices which are capable of performing reliable and sensitive assays in a cost-effective manner. The present application is directed to procedures which accomplish this goal.